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Method for treating pancreatitis with mesenchymal stem cells

a mesenchymal stem cell and pancreatitis technology, applied in the field of cell isolation, can solve the problems of high production cost, difficult identification, heterogeneity of cultured cells,

Inactive Publication Date: 2016-10-13
SCM LIFESCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020](v) administering the cells obtained in step (iv) to a subject suffering from pancreatitis, wherein the bone marrow, peripheral blood, cord blood, fatty tissue sample, or cytokine-activated peripheral blood cells do not undergo centrifugation of greater than 1,000 rpm in steps (i) to (iii), so as not to disturb the integrity of the cells or cause dissociation of cells from the tissue.

Problems solved by technology

Although it is difficult to identify whether these cells are distinct cell types due to lack of characteristic markers, they have some different level of surface marker expressions and various differentiation abilities, probably due to their distinct isolation and culture methods.
Potential disadvantages of these methods in terms of clinical applications are the heterogeneity of cultured cells, high risk of contamination, and / or high cost of production.
The isolation of MSCs by using the conventional gradient centrifugation method produces a heterogeneous population, which has led to confused results in the outcomes of clinical trials [41].

Method used

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  • Method for treating pancreatitis with mesenchymal stem cells
  • Method for treating pancreatitis with mesenchymal stem cells
  • Method for treating pancreatitis with mesenchymal stem cells

Examples

Experimental program
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example 1

Isolation of MLSCs and Cell Culture

[0151]One-ml of discarded human bone marrow aspirate, taken from the iliac crest of a patient undergoing bone marrow examination after informed consent and approval of the Inha University Medical School IRB, were mixed with 15 ml of complete growth medium: Dulbecco's modified Eagle's Medium (DMEM) containing high or low glucose (GIBCO-BRL, Life-technologies, MD USA), with 20% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and then incubated in 100 mm culture dish. As shown in FIG. 1, after 2 hour incubation at 37° C., 5% CO2, only the cell culture supernatant was transferred to a new dish. After another 2 hour incubation in a new dish, the supernatant was transferred to a non-coated, collagen or polylysine-coated dish and incubated for 2 hours (D1). After transferring the supernatant one more time to a new dish (D2), the supernatant was transferred to a new dish and then incubated for 1 day (D3). This was repeated two more times with 1 an...

example 2

Flow Cytometry Analysis

[0152]The isolated and expanded cells from single-cell derived colonies were characterized at passage 3 or 4 by flow cytometric analysis for a panel of cell surface proteins. The cells were harvested from 75 flask by treatment of trypsin / EDTA and washed with PBS twice. The cells were incubated in PBS with 0.1% goat serum for blocking and then washed with washing buffer (PBS with 0.4% BSA) twice. The cells were incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies for 40 min at 4° C. Tested antigens included matrix receptors (CD13, CD44, CD105), integrin (CD29), PECAM (CD31), ALCAM (CD166), SH3 and SH4 (CD73), Thy-1 (CD90) and hematopoietic lineage markers (CD34, HLA-DR, HLA-ClassI) (BD Biosciences Pharmingen, San Diego, Calif., USA). The cell mixture was then washed twice with washing buffer and analyzed using a fluorescence-activated cell sorter (FACS) with a 525 nm filter for green FITC fluorescence and with a 575 nm fi...

example 3

Induction of Multi-Lineage Differentiation

[0153]Pellet culture assay was used for chondrogenic, osteogenic, adipogenic differentiation experiments using passage 3 or 4 cells. 2×105 cells in 0.5 ml culture medium were spun down to make a pellet. The following supplements in DMEM containing high glucose and 20% FBS were used for each lineage, chondrogenic differentiation medium: 6.25 / ml insulin (Sigma Chemical Co, St Louis, Mo., USA), transferrin (Sigma), 6.25 ng / ml selenous acid (Sigma), 1.25 mg / ml BSA (Sigma), 5.35 / ml linoleic acid (Sigma), TGF-β1 10 ng / ml (R&D Systems, Minneapolis, Minn., USA), and TGF-β3 10 ng / ml (R&D Systems, Minneapolis, Minn., USA); osteogenic differentiation medium: 50 / ml ascorbate 2-phosphate (Sigma), 10−8 M dexamethasone (Sigma), and 10 mM β-glycerophosphate (Sigma); adipogenic differentiation medium: 50 / ml ascorbate 2-phosphate (Sigma), 10−7 M dexamethasone (Sigma), and 50 / ml indomethacine (Sigma). The pellet culture was incubated at 37° C., 5% CO2 and medi...

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Abstract

The present application discloses a method of treating pancreatitis by administering to the patient mesenchymal stem cells that are obtained by manipulating a biological sample of cells, which includes multi-lineage stem cells, progenitor cells, other marrow stromal cells: allowing the sample of cells to settle in a container; transferring supernatant from the container to another container; and isolating cells from the supernatant, which has comparatively lower density in the sample.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to the field of cell isolation. The present invention also relates to methods of isolating various types of stem cells or progenitor cells. The present invention also relates to a method of treating or alleviating pancreatitis by administering to the patient suffering from the condition the isolated stem cells or progenitor cells.[0003]2. General Background and State of the Art[0004]Bone marrow is known to contain hematopoietic and mesenchymal stem and progenitor cells. Hematopoietic stem cells (HSCs) can generate various types of blood cells [1], and marrow stromal cells (MSCs) or mesenchymal stem cells are capable of differentiating into several different tissues including cartilage, bone and adipose [2,3,4]. MSCs were first found by Friedenstein and his colleagues [5] based on their adherence to cell culture dish. Undifferentiated MSC are fibroblast-like in morphology, self-renewable, an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/18A61K35/12C12Q1/02A61P7/10A61P1/18A61K35/28A61P29/00A61K35/14A61K35/35A61K35/51
CPCA61K35/12A61K35/14A61K35/28A61K35/35A61K35/51A61P1/18A61P7/10A61P29/00A61K35/44
Inventor SONG, SUN UKLEE, DON-HAENGHONG, SOON-SUNJUNG, KYUNG HEE
Owner SCM LIFESCI CO LTD
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